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ALP Test Kit

(PNPP Method)
This reagent kit is intended for "in vitro" quantitative
determination of Alkaline Phosphatase activity in

Alkaline Phosphatase (ALP) is an enzyme of the Hydrolase
class of enzymes and acts in an alkaline medium. It is found in ALP activity (U/L) = DA/min. x 2764.
high concentrations in the liver, biliary tract epithelium and in the
bones. Normal levels are age dependent and increase during NORMAL VALUE : .
bone development. Increased levels are associated mainly with
Children (3-15 yrs) : 104 - 390 U/L
liver and bone disease. Moderate increases are seen in
Hodgkins disease and congestive heart failure. Adults : 25 - 140 U/L
Each Laboratory should establish it's own normal range
PRINCIPLE: representing its patient population.
Alkaline Phosphatase (ALP) at an alkaline pH hydrolases p-
Nitrophenylphosphate to form p-Nitrophenol and phosphate. LINEARITY :
The rate of formation of p-Nitrophenol is measured as an This procedure is linear upto 1600 U/L. If the absorbance change
increase in absorbance which is proportional to the ALP activity
(DA/min.) Exceeds 0.570, dilute the sample with normal saline (NaCl
in the sample.
0.9%) and repeat the assay.Multiply result by dilution factor.
P-NPP + HO PNP + PO4 For accuracy, it is advised to run known serum controls with each
Alk. medium assay.

Reagent 1 - AMP Buffer 1. Storage conditions as mentioned on the kit to be adhered.
Reagent 2 - PNPP Substrate 2. Do not freeze or expose the reagents to higher temperature as it
may affect the performance of the kit.
MATERIALS REQUIRED BUT NOT PROVIDED:- 3. Before the assay bring all the reagents to room temperature.
- Clean & Dry Glassware. 4. Avoid contamination of the reagent during assay process.
- Laboratory Glass Pipettes or Micropipettes & Tips. 5. Use clean glassware free from dust or debris.
- Bio-Chemistry Analyzer. 6. Reagent : sample ratio as mentioned here above must be strictly
observed as any change in to it will adversly effect the factor.
SAMPLES: 7.Donot use the reagent if the reagent is hazy or cloudy.
Unhaemolysed Serum, ALP is reported to be stable in serum for
3 days at 2 - 8°C. BIBLIOGRAPHY :
1.Rec. GSCC (DGKC) ; J. Clin. Chem. Clin. Biochem. 1972; 10 : 182.
PREPARATION OF REAGENT & STABILITY : 2.Heerspik. W., Hafkenscheidt J. C. M., Siepelvander Ven
Working Reagent : Jongekryg J., Djit C. C. M., Enzyme 25, 333 - 341 (1980).
Mix 4 part of Reagent - 1 with 1 part of Reagent - 2
Stability : 5 Days at 20 -25°C .30 days at 2-8°C.

Reaction type : Kinetic (Increasing) S05C 4 x 20 ml + 1 x 20 ml 4 x 20 ml 1 x 20 ml
Wave Iength : 405 nm
Temperature : 37°C IVD
Delay : 60 sec. UNIONPROJET S.R.L.S.

Interval : 60 sec. EC REP Via Aliprandi 41-20851
Lissone (MB)

ISO 9001:2015 ISO 13485:2003

No.of reads :3
Reagent volume : 1.0 ml
Sample volume : 20 µl
Factor : 2764
Zero setting : Deionosed water
Light path : 1 cm

Pipette into clean dry test tube labelled as Test (T) :
Addition Sequence (T)
Working Reagent 1.0 ml
Sample 20 µl

Mix well and read the initial absorbance A0after 1 minute &
repeat the absorbance reading after every 1, 2, & 3 minute.
Calculate the mean absorbance change per minute (D A/min).

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